Photobiomodulation in the infrared spectrum reverses the expansion of circulating natural killer cells and brain microglial activation in Sanfilippo mice.

Sanfilippo syndrome results from inherited mutations in genes encoding lysosomal enzymes that catabolise heparan sulfate (HS), leading to early childhood-onset neurodegeneration. This study explores the therapeutic potential of photobiomodulation (PBM), which is neuroprotective and anti-inflammatory in several neurodegenerative diseases; it is also safe and PBM devices are readily available. We investigated the effects of 10-14 days transcranial PBM at 670 nm (2 or 4 J/cm2/day) or 904 nm (4 J/cm2/day) in young (3 weeks) and older (15 weeks) Sanfilippo or mucopolysaccharidosis type IIIA (MPS IIIA) mice. Although we found no PBM-induced changes in HS accumulation, astrocyte activation, CD206 (an anti-inflammatory marker) and BDNF expression in the brains of Sanfilippo mice, there was a near-normalisation of microglial activation in older MPS IIIA mice by 904 nm PBM, with decreased IBA1 expression and a return of their morphology towards a resting state. Immune cell immunophenotyping of peripheral blood with mass cytometry revealed increased pro-inflammatory signalling through pSTAT1 and p-p38 in NK and T cells in young but not older MPS IIIA mice (5 weeks of age), and expansion of NK, B and CD8+ T cells in older affected mice (17 weeks of age), highlighting the importance of innate and adaptive lymphocytes in Sanfilippo syndrome. Notably, 670 and 904 nm PBM both reversed the Sanfilippo-induced increase in pSTAT1 and p-p38 expression in multiple leukocyte populations in young mice, while 904 nm reversed the increase in NK cells in older mice. In conclusion, this is the first study to demonstrate the beneficial effects of PBM in Sanfilippo mice. The distinct reduction in microglial activation and NK cell pro-inflammatory signalling and number suggests PBM may alleviate neuroinflammation and lymphocyte activation, encouraging further investigation of PBM as a standalone, or complementary therapy in Sanfilippo syndrome.

Indirect application of near infrared light induces neuroprotection in a mouse model of parkinsonism - an abscopal neuroprotective effect.

We have previously shown near infrared light (NIr), directed transcranially, mitigates the loss of dopaminergic cells in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice, a model of parkinsonism. These findings complement others suggesting NIr treatment protects against damage from various insults. However one puzzling feature of NIr treatment is that unilateral exposure can lead to a bilateral healing response, suggesting NIr may have 'indirect' protective effects. We investigated whether remote NIr treatment is neuroprotective by administering different MPTP doses (50-, 75-, 100-mg/kg) to mice and treating with 670-nm light directed specifically at either the head or body. Our results show that, despite no direct irradiation of the damaged tissue, remote NIr treatment produces a significant rescue of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta at the milder MPTP dose of 50-mg/kg (∼30% increase vs sham-treated MPTP mice, p<0.05). However this protection did not appear as robust as that achieved by direct irradiation of the head (∼50% increase vs sham-treated MPTP mice, p<0.001). There was no quantifiable protective effect of NIr at higher MPTP doses, irrespective of the delivery mode. Astrocyte and microglia cell numbers in substantia nigra pars compacta were not influenced by either mode of NIr treatment. In summary, the findings suggest that treatment of a remote tissue with NIr is sufficient to induce protection of the brain, reminiscent of the 'abscopal effect' sometimes observed in radiation treatment of metastatic cancer. This discovery has implications for the clinical translation of light-based therapies, providing an improved mode of delivery over transcranial irradiation.

Some certainty for the "zone of uncertainty"? Exploring the function of the zona incerta.

The zona incerta (ZI), first described over a century ago by Auguste Forel as a "region of which nothing certain can be said," forms a collection of cells that derives from the diencephalon. To this day, we are still not certain of the precise function of this "zone of uncertainty" although many have been proposed, from controlling visceral activity to shifting attention and from influencing arousal to maintaining posture and locomotion. In this review, I shall outline the recent advances in the understanding of the structure, connectivity and functions of the ZI. I will then focus on a possible and often neglected global role for the ZI, one that links its diverse functions together. In particular, I aim to highlight the idea that the ZI forms a primal center of the diencephalon for generating direct responses (visceral, arousal, attention and/or posture-locomotion) to a given sensory (somatic and/or visceral) stimulus. With this global role in mind, I will then address recent results indicating that abnormal ZI activity manifests in clinical symptoms of Parkinson disease.

Organisation of connections between the zona incerta and the interposed nucleus.

We have examined the organisation of connections between the zona incerta (ZI), a small diencephalic nucleus deriving from the ventral thalamus, and the interposed nucleus (Int) of the cerebellum. Injections of the tracer cholera toxin subunit B were made into either the ZI or Int of Sprague Dawley rats by using stereotaxic coordinates. We have two major findings. First, there is a heavy projection from Int to ZI; there is also a small projection back to Int from ZI. After injections into Int, labelled terminals and cells tend to concentrate within the medial region of each of the cytoarchitectonically defined sectors of ZI. Second, there is an unusual laterality of connectivity between the ZI and the Int. The projection from the Int to the ZI is mainly contralateral, whilst the ZI projection back to the Int is mainly ipsilateral. In conclusion, our results indicate that the Int of the cerebellum provides a rich source of afferents to the ZI, rendering the latter in a key position to integrate information from the Int together with many other types of subcortical information it receives, particularly from the brainstem.

Zona incerta: Substrate for contralateral interconnectivity in the thalamus of rats.

We have shown previously that the zona incerta (ZI), a small nucleus deriving from the ventral thalamus, has extensive ipsilateral connections with the higher order and intralaminar nuclei of the dorsal thalamus and that there are many ipsilateral interconnections between the different cytoarchitectonic sectors of the ZI. In this study, we explore the contralateral connections that the ZI has with its opposing nucleus as well as with the other nuclei of the thalamus. Injections of biotinylated dextran or cholera toxin subunit B were made into each of the different ZI sectors (rostral, dorsal, ventral, and caudal) and into intralaminar and higher order dorsal thalamic nuclei of Sprague-Dawley rats by using stereotaxic coordinates. Brains were fixed in aldehyde and processed using standard methods. Our results show that, after injections limited to a given ZI sector, labelled terminal-like elements and cells were seen across the other sectors of the ZI of the contralateral side. Furthermore, after each of these ZI injections, labelling was seen in the intralaminar (e.g., parafascicular, central lateral, and central medial) and higher order (e.g., posterior thalamic, lateral posterior, and lateral dorsal) nuclei of the contralateral side. These patterns of labelling were confirmed after tracer injections into intralaminar and higher order nuclei; after such injections, labelling was seen in the contralateral ZI. In all cases, there was labelling on the ipsilateral side as well, and this was generally heavier than on the contralateral side. Overall, our results indicate that there is a network of interconnections between the ZI of both sides of the thalamus and that the ZI has contralateral connections with the intralaminar and higher order nuclei. Hence, the ZI furnishes a substrate that spreads activity to both sides of the brain.

Evidence for a visual subsector within the zona incerta.

Here we examine the patterns of connections between the zona incerta (ZI) of the thalamus and the major visual centers of the rat brain, namely the retina, dorsal lateral geniculate nucleus (LGd), superficial layers of the superior colliculus (SCs), and occipital cortex (Ocl). Injections of the tracers biotinylated dextran or cholera toxin subunit b were made into each of these centers, as well as ZI itself, by using stereotaxic coordinates. Rat brains were then aldehyde-fixed and processed using standard methods. We show that the retina, LGd, SCs, and Ocl all have connections with ZI; moreover, that each of these connections make a very distinct territory or subsector within the most lateral ZI regions. This subsector of connectivity with the visual centers does not respect the well-defined cytoarchitectonic sectors of ZI, being made up of small zones in the dorsal, ventral, and caudal sectors. Often, a distinctive "horse-shoe" pattern is evident, particularly after retinal and Ocl injections. Tracer injections into topographically distinct regions of the LGd. SCs, or Ocl results in no shift in the spatial location of labelling within ZI; after each injection, labelling is always seen within the lateral edge of the nucleus. Labelled terminals and cells are seen after LGd and SCs injections, while only labelled terminals are seen after retinal and Ocl injections. Although the precise function of this novel visual subsector is not known, these early findings suggest that ZI may be in a position to integrate visual information together with the other somatosensory, motor, and visceral information that it receives.

Induction of Fos-like immunoreactivity in the ventral thalamus after electrical or chemical stimulation of various subcortical centres of rats.

We have examined the patterns of Fos-like immunoreactivity in the ventral thalamus (thalamic reticular nucleus (Rt), zona incerta (ZI) and ventral lateral geniculate nucleus (LGv)) after electrical or chemical stimulation of nuclei in either the brainstem (midbrain reticular nucleus), basal forebrain (substantia innominata) or dorsal thalamus (parafascicular nucleus). Sprague-Dawley rats were anaesthetised with Halothane or Ketamil/Rompun and the above mentioned centres were stimulated either electrically or chemically (using kainic acid). Brains were then processed for Fos-like immunocytochemistry using standard methods. We detected no major differences in the labelling patterns after either electrical or chemical stimulation or after using Halothane or Ketamil/Rompun anaesthesia. After brainstem or dorsal thalamic stimulations, many Fos-like immunoreactive cells were seen within the rostral pole of the Rt, the dorsal sector of the ZI and the parvocellular lamina of the LGv. After basal forebrain stimulations, many Fos-like immunoreactive cells were seen in the rostral pole of the Rt and rostral sector of the ZI, but very few were apparent in the LGv. Overall, our results show that distinct groups of cells in the ventral thalamus show increased levels of Fos-like immunoreactivity after stimulation of different subcortical centres. These activated cells of the ventral thalamus, are in turn, in a position to influence particular thalamocortical pathways through their dorsal thalamic projections.

Lamination of spinal cells projecting to the zona incerta of rats.

In this study, the lamination patterns of spinal cells projecting to the zona incerta (ZI), intralaminar nuclei and ventral posterior nucleus of the thalamus have been explored. Injections of cholera toxin subunit B or latex beads were made into the ZI, intralaminar and ventral posterior nuclei of Sprague Dawley rats. The brain and spinal cord were then aldehyde fixed and processed using standard methods. Our results show two major findings. First, after injections into the ZI, there is a distinct pattern of lamination of labelled cells in the spinal cord, a pattern that changes across the different levels. At cervical levels, labelled cells are located within the medial region of the deep dorsal horn, while at lumbar and sacral levels, they are found in the intermediate grey matter. These results are similar to those seen after injections into the intralaminar or ventral posterior nuclei, except that in the latter cases, more labelled cells are located in the superficial laminae of the dorsal horn, particularly from the ventral posterior nucleus. Second, the ZI is not associated uniformly with all spinal levels; labelling is heaviest at cervical and lightest at thoracic levels. From each thalamic injection site, labelling is noted on both sides of the spinal cord, with a clear contralateral predominance. In conclusion, the results indicate that the ZI receives a distinct set of spinal projections principally from the cervical level. The particular pattern of lamination of spinal cells projecting to the ZI suggests that the type of information relayed is from deep somatic and/or visceral structures, and probably nociceptive in nature.

Dorsal thalamic connections of the ventral lateral geniculate nucleus of rats.

In order to understand better the organisation of the ventral lateral geniculate nucleus of the ventral thalamus, this paper has examined the patterns of connections that this nucleus has with various nuclei of the dorsal thalamus in rats. Injections of biotinylated dextran or cholera toxin subunit B were made into the parafascicular, central lateral, posterior thalamic, medial dorsal, lateral dorsal, lateral posterior, dorsal lateral geniculate, anterior, ventral lateral, ventrobasal and medial geniculate nuclei of Sprague-Dawley rats and their brains were processed using standard tracer detection methods. Three general patterns of ventral lateral geniculate connectivity were seen. First, the parafascicular, central lateral, medial dorsal, posterior thalamic and lateral dorsal nuclei had heavy connections with the parvocellular (internal) lamina of the ventral lateral geniculate nucleus. This geniculate lamina has been shown previously to receive heavy inputs from many functionally diverse brainstem nuclei. Second, the visually related dorsal lateral geniculate and lateral posterior nuclei had heavy connections with the magnocellular (external) lamina of the ventral lateral geniculate nucleus. This geniculate lamina has been shown by previous studies to receive heavy inputs from the visual cortex and the retina. Finally, the anterior, ventral lateral, ventrobasal and medial geniculate nuclei had very sparse, if any, connections with the ventral lateral geniculate nucleus. Overall, our results strengthen the notion that one can package the ventral lateral geniculate nucleus into distinct visual (magnocellular) and non-visual (parvocellular) components.

Organization of brain stem afferents to the ventral lateral geniculate nucleus of rats.

We have examined the patterns of projections from different nuclei of the brain stem to the ventral lateral geniculate nucleus (vLGN) of the thalamus. Injections of biotinylated dextran were made into different nuclei of the brainstem (i.e., midbrain reticular nucleus, pontine reticular nucleus, deep layers of superior colliculus, periaqueductal grey matter [ventrolateral, dorsolateral, and lateral columns], pedunculopontine tegmental nucleus, parabrachial nucleus, lateral dorsal tegmental nucleus, substantia nigra [pars reticulata], locus coeruleus, and dorsal raphe) of Sprague-Dawley rats using stereotaxic coordinates. Our results show that all of the above mentioned brain-stem nuclei have overlapping projections to the medial regions of the vLGN, within the parvocellular lamina of the nucleus. This if the first instance of the parvocellular lamina being shown to receive a major set of projections. Very few labelled terminals from the brain stem were ever seen within the larger more lateral magnocellular lamina, which has been shown by previous studies to receive heavy inputs from visually associated structures, such as the retina and occipital cortex. Since many of the brain-stem nuclei injected in this study have little to do with visual processing, our results suggest that one can perhaps package the vLGN into distinct visual (magnocellular) and nonvisual (parvocellular) components.

Organisation of the amygdalo-thalamic pathways in rats.

This study examines the organisation of the pathways from the amygdala to the thalamus. Amygdaloid nuclei (medial, central, basolateral and olfactory groups) of Sprague-Dawley rats were injected with biotinylated dextran using stereotaxic coordinates and their brains were then aldehyde-fixed and processed using standard methods. We have three major findings. First, the amygdala has a distinct set of projections to particular nuclei of the thalamus. The thalamic nuclei with the heaviest amygdaloid terminations include the zona incerta, the mediodorsal and the midline nuclei. Second, nuclei of different amygdaloid groups project to the thalamus in slightly different patterns. For example, some groups of nuclei project to the thalamic reticular nucleus (e.g. medial, olfactory) whilst others do not (e.g. central, basolateral). Thus, there is a certain amount of heterogeneity within the amygdaloid projections to the thalamus. Third, when we compare our results on the amygdalo-thalamic pathways to the many previous descriptions of the thalamo-amygdaloid pathways, we note that they are largely out of register. In other words, some of the thalamic nuclei that project to a given group of amygdaloid nuclei do not necessarily receive a projection back from that same amygdaloid nucleus. Hence, there is no substrate for a strong feed-back relationship between the thalamus and the amygdala, as there has been shown for other centres of the brain (e.g. between the thalamus and neocortex).

Does the perireticular thalamic nucleus project to the neocortex?

This study defines several features of the early connections of the developmentally transient perireticular thalamic nucleus of rats. The neocortex of developing rats was injected with either DiI, biotinylated dextran, WGA-HRP (wheatgerm agglutinin conjugated-horseradish peroxidase), fluorescent latex beads or cholera toxin subunit B (CTB) and their brains were processed for tracer detection with standard methods. In general, tracer injections into various regions of the developing neocortex revealed no labelled neurones within the perireticular nucleus, although some of these tracers (WGA-HRP, dextran) labelled many of the amoeboid microglial cells that are found within this nucleus. There were, however, many retrogradely labelled neurones in a region adjacent to the perireticular nucleus, within the nucleus basalis of the basal forebrain (medial edge of globus pallidus). Their identity was confirmed as neurones of the nucleus basalis since they were all were similar in morphology and somal size to neurones that were immunoreactive to NGFr (nerve growth factor receptor), an antigen found only among neurones of the nucleus basalis and basal forebrain. Moreover, double labelling experiments revealed that most, if not all, of the cortically labelled neurones were NGFr-immunoreactive also. Thus, in conclusion, our results suggest that the perireticular nucleus does not project to the neocortex; the only neurones in the general vicinity of the perireticular nucleus that have a cortical projection form part of the nucleus basalis.

Organization of the basal forebrain projection to the thalamus in rats.

We have examined the patterns of projections that exist between the different nuclei of the basal forebrain (BF) and the thalamus. Injections of biotinylated dextran were made into different nuclei of the BF (i.e. substantia innominata, nucleus basalis of Meynert, vertical and horizontal limbs of the diagonal band) of Sprague-Dawley rats using stereotaxic coordinates. Our results show that all of the above-mentioned BF nuclei have projections to the thalamus and that projections from different nuclei are rather similar. The bulk of the BF afferents terminate within the intralaminar, midline and mediodorsal nuclei of the dorsal thalamus and the zona incerta and reticular nucleus of the ventral thalamus. Very few terminals are ever seen in the other nuclei of the thalamus. Thus our results indicate that the BF targets particular thalamic nuclei, thereby being in a position to influence distinct thalamo-cortical pathways.

Organisation of the cortical projection to the zona incerta of the thalamus.

In this study, we examined the organisation of the cortical projection to a small nucleus of the ventral thalamus called the zona incerta. Injections of biotinylated dextran were made into various cytoarchitectonically defined cortical areas of Sprague-Dawley rats, including the frontal (Fr1), cingulate (Cg1), parietal (Par1), forelimb (FL), and occipital (Oc1), and the patterns of anterograde labelling were examined in the zona incerta. In addition, injections of cholera toxin subunit B (CTB) were made into the zona incerta itself, and the laminar locations of retrogradely labelled cells were examined. Brains were then fixed in aldehyde and processed by using standard methods. After injections of dextran into the different cortical areas, the distribution and number of labelled terminals in the zona incerta were quite different. In terms of number, Cg1 had the heaviest projection to the zona incerta, whereas Oc1 had the weakest. In terms of distribution, three distinct patterns were evident. Firstly, labelled terminals were seen in distinct clusters in the dorsal and ventral sectors, usually one overlying the other (Fr1, FL, Par1). Secondly, labelled terminals were seen occupying much broader zones in the dorsal (and rostral) sector (Cg1). Thirdly, very few labelled terminals were seen in any incertal sector (Oc1). Thus, these results suggest that not all cortical areas project to the zona incerta equally, with some areas having stronger projections to this nucleus than others. From our CTB injections into the zona incerta itself, we show that all retrogradely labelled cells in the neocortex were limited to layer V. We suggest a possible thalamic circuit involving the zona incerta, the thalamic reticular nucleus (the other major nucleus of the ventral thalamus), and the two types of corticothalamic afferents (from layer VI and V).

Evidence for extensive inter-connections within the zona incerta in rats.

We have examined whether there is an extensive system of inter-connections between the functionally distinct sectors of the zona incerta (ZI) of the thalamus. Unilateral injections of biotinylated dextran were made into each of the different incertal sectors (rostral, dorsal, ventral and caudal) of Sprague-Dawley rats by using stereotaxic coordinates. Our results show that after separate injections limited to each of the incertal sectors, many labelled terminals and cells were seen across the different sectors of the ipsilateral, as well as the contralateral side, with the heaviest labelling being on the side ipsilateral to the injection. Thus, these results suggest that the ZI is in a position to integrate an extensive array of afferents from many functionally diverse neural centres of the brain.

Distribution of various neurochemicals within the zona incerta: an immunocytochemical and histochemical study.

To gain insight into the cellular organisation of the zona incerta, we have examined the chemoarchitectonic properties of this "uncertain zone". The brains of Sprague-Dawley rats and common cats were processed for immunocytochemistry or NADPH-diaphorase histochemistry using standard methods. For the immunocytochemistry, antibodies to y-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), parvalbumin, calbindin, tyrosine hydroxylase, somatostatin, serotonin and glutamate were used. Two general patterns of distribution in the zona incerta were seen. First, labelled cells were restricted largely to one of the cytoarchitectonically defined sectors of the zona incerta. For instance, GABA, GAD and parvalbumin-immunoreactive cells were found principally within the ventral sector, NADPH-diaphorase and glutamate-immunoreactive cells within the dorsal sector and tyrosine hydroxylase- and somatostatin-immunoreactive cells within the rostral sector. Second, labelled cells were scattered somewhat across all incertal sectors, with no clear region of concentration. This pattern included the calbindin- and serotonin-immunoreactive cell groups. These results indicate that the zona incerta is made up of many neurochemically distinct cell groups, some of which respect the well-defined cytoarchitectonic boundaries of the nucleus, whilst others do not. This rich neurochemical diversity in the zona incerta suggests that this nucleus may have differential effects on the different structures that it projects to.

Evidence for a large projection from the zona incerta to the dorsal thalamus.

In an effort to understand better how the zona incerta may influence neocortical activity, this study has examined the patterns of projection that this nucleus has to the dorsal thalamus, the "gateway" to the neocortex. To this end, Sprague-Dawley rats were anaesthetised with Ketamil (100 mg/kg) and Rompun (10 mg/kg), and injections of biotinylated dextran or cholera toxin subunit B (CTB) were made into various dorsal thalamic nuclei, including the primary relay (dorsal lateral geniculate, medial geniculate, ventral posterior), association (lateral dorsal, lateral posterior, posterior thalamic), and intralaminar (central lateral, parafascicular) nuclear groups, by using stereotaxic coordinates. Brains were aldehyde fixed and processed with standard methods. Our results show that there is a large projection from the zona incerta to the dorsal thalamus. This projection does not blanket all nuclei of the dorsal thalamus but, rather, shows a clear preference for some nuclei over others. After CTB or dextran injections into the primary relay nuclei, very few cells are labelled in the zona incerta. After similar injections are made into the association or intralaminar nuclei, however, many more labelled incertal cells are seen. There are some differences in the distribution of labelled cells within the zona incerta after injections into the association nuclei compared with injections into the intralaminar nuclei. The association nuclei relate strongly to the ventral sector, whereas the intralaminar nuclei relate strongly to the dorsal sector of the zona incerta. After each of these injections into the dorsal thalamus, labelled terminals are seen in the zona incerta also, and their distribution mirrors the distribution of the labelled incertal cells described above. Thus, in summary, our results indicate that the zona incerta has a large and preferential projection to the dorsal thalamus, in particular from the association and intralaminar nuclei. Through this dorsal thalamic projection, the zona incerta is in a position to influence large areas of the neocortex.

Specificity of projection among cells of the zona incerta.

We have examined whether individual cells of the zona incerta of the thalamus have widespread projections across the brain. Double injections of different coloured fluorescent latex beads (red or green) were made, in various combinations, into regions of neocortex, dorsal thalamus or brainstem of Sprague-Dawley rats. These regions were chosen since they have been shown previously to receive projections from the zona incerta. We also made injections of different coloured beads into different regions of these same brain centres (ie, distinct cortical areas or individual dorsal thalamic and brainstem nuclei). In general, our results show that cells of the zona incerta have projections limited to one of these brain centres only. We saw very few double-labelled incertal cells after double injections of different coloured latex beads into either the neocortex/dorsal thalamus, neocortex/brainstem or dorsal thalamus/brainstem. Further, we show that within each of these brain centres, the projection patterns of individual incertal cells is rather restricted, since double injections of different coloured beads into separate regions of the same centre resulted in few double-labelled incertal cells. Taken together, these results suggest a very clear specificity of projection among cells of the zona incerta. Thus, although the cells of the zona incerta receive a plethora of inputs from many sources, it appears that its cells have a very clear and focussed output to distinct regions of the brain.

Patterns of brainstem projection to the thalamic reticular nucleus.

To understand better how the brainstem may influence thalamocortical activity, we have examined the projection patterns of different brainstem nuclei to the thalamic reticular nucleus. Iontophoretic injections of biotinylated dextran were made into various nuclei of the brainstem (superior colliculus, periaqueductal grey matter, parabrachial nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, substantia nigra, ventral tegmental area, and locus coeruleus) of Sprague-Dawley rats by using stereotaxic coordinates. Our results show that afferents from each brainstem nucleus make distinct zones within the reticular nucleus. For example, the superior colliculus projects largely to the dorsal parts of the reticular nucleus, whereas the pedunculopontine nucleus projects to the ventral parts of the reticular nucleus. The substantia nigra, on the other hand, projects to the ventrolateral edge of the reticular nucleus. We also examined the distribution of these brainstem afferents within the dorsal thalamus and compared these distributions with those seen in the reticular nucleus. We found three different patterns. First, a given brainstem nucleus projects to a particular dorsal thalamic nucleus as well as to the corresponding, functionally associated, reticular sector (e.g., from the substantia nigra). Second, a given brainstem nucleus projects to a particular dorsal thalamic nucleus but not to the corresponding reticular sector (e.g., from the superior colliculus). Finally, a given brainstem nucleus projects to a given reticular sector but not to the corresponding dorsal thalamic nucleus (e.g., from the midbrain reticular nucleus). In general, our results indicate that various brainstem nuclei project to particular territories of the thalamic reticular nucleus. Through these reticular projections, brainstem nuclei may influence distinct thalamocortical pathways in addition to those that are influenced by their direct projection to the dorsal thalamus.

Patterns of connections between zona incerta and brainstem in rats.

To understand better the organisation of zona incerta of the thalamus, this study has examined the patterns of connections that this nucleus has with various nuclei of the brainstem. Injections of biotinylated dextran or cholera toxin subunit B were made into the dorsal raphe, midbrain reticular nucleus, pedunculopontine tegmental nucleus, periaqueductal grey matter, pontine reticular nucleus, substantia nigra, superior colliculus, and ventral tegmental area of Sprague-Dawley rats, and their brains were processed by using standard tracer-detection methods. In general, our results show that zona incerta forms the major zone in the thalamus where these ascending brainstem axons terminate and from which descending axons that travel back to these same brainstem centres originate. These incertal inputs and outputs are limited largely to a distinct sector of zona incerta, the dorsal sector. An exception to this pattern is evident in the incertal projection to the deep layers of the superior colliculus; this projection, unlike all of the others, arises from cells in the ventral sector of zona incerta. Our results also show little evidence for a well-defined topography of projection between the brainstem and the zona incerta. For instance, small injections into each brainstem nucleus result in labelled terminals and in cells spread throughout much of the dorsal sector of zona incerta, with no local zone of concentration within the sector. Again, an exception to this pattern is seen in the incertal projection to the superior colliculus. This projection, unlike the others, shows a clear topographical organisation: A medial-lateral shift in the injection site in the colliculus results in a lateral-medial shift in the position of labelled cells in zona incerta. Curiously, even though the incertal projection to the colliculus appears to be mapped, the collicular projection back to zona incerta is not mapped. In conclusion, then, a number of brainstem nuclei (except for the deep collicular layers) have strong and overlapping connections within the same sector of zona incerta. This convergence of many functionally diverse brainstem afferents within zona incerta places this nucleus in a strategic position to sample the general activity of the brainstem and, perhaps, acts as a relay of this information to higher centres, such as the dorsal thalamic relay nuclei and the cerebral hemispheres.